Suarez P, Zardoya R, Prieto C, et al.: 1994, Direct detection of the porcine reproductive and respiratory syndrome (PRRS) virus by reverse polymerase chain reaction (RT-PCR). Van Woensel[r]
(1)Detection of porcine reproductive and respiratory syndrome virus by reverse transcription–polymerase chain reaction using
different regions of the viral genome
Helena Guarino, Sagar M Goyal, Michael P Murtaugh, Robert B Morrison, Vivek Kapur Abstract. Serologic studies have revealed strain variability between American and European isolates and among American isolates of porcine reproductive and respiratory syndrome virus (PRRSV) The objective of this study was to develop an assay for the routine diagnosis of PRRSV in field specimens using reverse transcription–polymerase chain reaction (RT-PCR) amplification of conserved genomic regions Twenty-four field isolates of PRRSV from different regions of the USA were analyzed in the study Six primer pairs from open reading frames (ORFs) 4, 6, and of the American strain (ATCC VR-2332) and from ORF 1b of the Lelystad strain were used for the amplification of the viral genome by PCR Amplification products of the expected sizes were obtained from all isolates by PCR amplification of ORF 7, the gene encoding the nucleo-capsid protein Oligonucleotide primers designed to amplify ORFs and detected 92% and 96% of the isolates, respectively, whereas primers for the amplification of ORF 1b detected 88% of all isolates The specificity of the amplified products of ORF from field isolates and reference strains was confirmed by chemiluminescent hybridization using an internal digoxigenin-labeled DNA probe Sequence analysis of this region indicated variation in the nucleotide sequence of isolates that did not hybridize with the internal probe These results indicate that ORF may serve as a potential target for the detection of PRRSV strains by RT-PCR and that genomic variability should be considered when nucleic acid hybridization is used to confirm the specificity of PCR amplification for diagnostic purposes.
Porcine reproductive and respiratory syndrome
(PRRS) was first reported in 1987 in the USA12 and
has subsequently been recognized throughout Europe
and Southeast Asia, including Japan.7,27,31 The disease
is caused by PRRS virus (PRRSV), which is a small, enveloped RNA virus belonging to the arterivirus group, which also includes equine arteritis virus, lac-tate dehydrogenase elevating virus, and simian
hem-orrhagic fever virus.20 The complete nucleotide
se-quence of the European strain of PRRSV (Lelystad virus) and partial sequences of the American strain
(ATCC VR-2332) have been determined.5,13–15,17–19,22,23
The genome of PRRSV consists of open reading frames (ORFs), of which are expressed by the
for-mation of subgenomic RNAs.19 ORFs 1a and 1b
en-code the viral RNA polymerase, and the remaining 6 ORFs encode small polypeptides consisting of 128–
265 amino acids each.6 ORFs 2–6 encode structural
proteins, and ORF appears to encode a nucleocapsid protein.19
Serologic studies have revealed strain variability among PRRSV isolates, mostly between the American and the European strains but also among American
From the Departments of Veterinary Diagnostic Medicine (Guar-ino, Goyal), Veterinary Pathobiology (Murtaugh, Kapur), and Clin-ical and Population Sciences (Morrison), College of Veterinary Med-icine, University of Minnesota, St Paul, MN 55108
Received for publication May 8, 1997
isolates.3,14,25,30,34 The antigenic variation between
American isolate VR-2332 and the Lelystad virus was
correlated with significant sequence differences,23and
it has been suggested that American and European
strains may represent distinct genotypes of PRRSV.21
Polymerase chain reaction (PCR), a sensitive and specific assay for the diagnosis of many infectious dis-eases, has recently been described for the detection of
PRRSV in clinical samples, including boar semen.4,28,29
One of the critical parameters in the PCR technique is the selection of the primer pairs, because nucleotide
mismatch could lead to false negative results.10In the
present study, we analyzed 24 different field isolates of PRRSV by single and nested PCR using primer pairs from different regions of the virus genome The object was to identify universal primers to amplify the genome of all or most PRRSV isolates The specificity of PCR products from ORF was determined by hy-bridization with a nonradiolabeled oligonucleotide probe.
Materials and methods
Virus isolates Twenty-four isolates of PRRSV were
ob-tained from 10 different states in the USA (Table 1) These viruses were isolated from either lungs or sera of clinically affected pigs in primary porcine alveolar macrophages or in CL 2621 cells by previously described methods.3The
(2)Table 1. Source of PRRSV isolates
Location Isolate number*
Illinois Indiana Iowa Kansas Kentucky
IL-4, IL-6 IN-4
IA-1, IA-2, IA-3, IA-15, IA-16 KS-1, KS-2
KY-2
Minnesota MN-1, MN-3, MN-4, SG-1,
SG-3, SG-4, SG-5, HL, WL Missouri
Nebraska Ohio South Dakota
MO-2 NE-1 OH-1 SD-1
* In addition to the above, ATCC VR-2332 and the Lelystad virus were used as reference strains of PRRSV
Table 2. Oligonucleotide primer sets
Primer set Primer sequence (59to 39)
Primer location (bp)
Predicted PCR product (bp) Single PCR (sense/antisense)
P41/42 GACGGCGGCAATTGGTTTC
GCAATCGCGAGCAACAGCC
1,227–1,245 1,842–1,824
615
P71/72 GCTGTTAAACAGGGAGTGG
CGCCCTAATTGAATAGGTGAC
2,867–2,885 3,375–3,355
508
P61/431 GTTTCAGCGGAACAATGGG
GCTGATTGACTGGCTGGCC
2,371–2,389 2,964–2,946
593
PR1b-P1 GACCCCGTCACCAGTGTGTC
GTCCGTTCTGAAACCCAGCA
8,748–8,767 9,005–8,986
257 Nested PCR
8214/8215 TCGTGTTGGGTGGCAGAAAAGC
GCCATTCACCACACATTCTTCC
2,763–2,785 3,247–3,225
484
8216/8217 CCAGATGCTGGGTAAGATCATA
CAGTGTAACTTATCCTCCCTGA
2,885–2,907 3,121–3,099
236
P61/72 GTTTCAGCGGAACAATGGG
CCCTAATTGAATAGGTGAC
2,371–2,389 3,375–3,355
1,004
P71/431 GCTGTTAAACAGGGAGTGG
GCTGATTGACTGGCTGGCC
2,867–2,885 2,964–2,946
97 modified serum neutralization test33was used to titrate these
isolates; the titers ranged from 102to 107.5TCID 50/ ml.
Reference strains The American (ATCC VR-2332) and
the European (Lelystad) strains of PRRSV at passages 10 and 7, respectively, were used as reference virus strains.
RNA isolation RNA was extracted from virus isolates
using a commercial silica-based membrane kit.aThe samples
(140 ml) were mixed with lysis buffer containing carrier RNA to facilitate the extraction of RNA from the samples. After incubation for 10 at room temperature, the sam-ples were precipitated with ethanol, added to a spin column, and centrifuged at 6,000 3 g for The column was
then washed twice to eliminate contaminants, and RNA was eluted in 50 ml of DEPC-treated water and kept at270 C until use Mock-infected cell culture fluid was used as a neg-ative control.
Effect of virus suspension in serum To determine if PCR
could detect PRRSV present in porcine sera, of 24 field isolates and the reference strains were diluted 1:5 in a
porcine serum known to be negative for PRRSV and its an-tibody Viral RNA from these virus-containing serum sam-ples was extracted and amplified by PCR.
Synthesis of cDNA Reverse transcription (RT) of RNA
was carried out in a 20-ml reaction volume containing ex-tracted RNA and 2.5mM of random hexamers.b The RNA
was denatured at 65 C for and then cooled on ice. Master mix consisting of 13PCR buffer, mM MgCl2,2.5
U/ml of murine leukemia virus reverse transcriptase,b1 U/ml
of RNAse inhibitor, and mM each of dATP, dCTP, dTTP, and dGTP was added at 16ml/sample The tubes were kept at room temperature for 10 to allow extension of the primers and then incubated in a thermal cycler (GeneAmp PCR system 9600)bat 42 C for 15 min, 99 C for min, and
5 C for and then stored at C.
PCR primers and probe The nucleotide sequences of
ORFs 4, 6, and of the American strain VR-2332 and ORF 1b of the Lelystad strain were used to select primers for single PCR These oligonucleotides were designed to am-plify several regions of the PRRSV genome (Table 2) In addition to single PCR, two different nested PCR reactions were also evaluated The design of the primer pairs used for nested PCR-1 (outer 8214/8215 and inner 8216/8217) and the internal probe (59-TGTCAGACATCACTTTACCC-39, nucleotides 3002–3022) was based on ORF of strain VR-2332.c,dNested PCR-2 was performed using P61/P72 as
out-er primout-ers and 431P/P71 as innout-er primout-ers The primout-er se-quences, their location in the genome, and the expected frag-ment size of PCR products for each primer set are shown in Table 2.
PCR reactions For single PCR reactions and for the first
stage of the nested PCR, cDNA was added to a mixture of 2 mM MgCl2,13 PCR buffer, 0.4 mM of each primer, and
0.5 U of Amplitaq DNA polymeraseb in a total volume of
(3)Figure 1. Agarose gel electrophoresis of PRRSV reference strain ATCC VR-2332 using primers from different ORFs Lane 1: P41/P42 (615 bp); lane 2: P71/P72 (508 bp); lane 3: P61/P431 (593 bp); lane 4: LV-1b (257 bp); lane 5: P14/P15 (484 bp); lane 6: P16/ P17 (236 bp); lane 7: P61/P72 (1,004 bp); lane 8: P431/P71 (97 bp); lane 9: negative control (PCR mix); MM: molecular weight marker fX174 RF DNA HaeIII.
55 C for 30 sec, and 72 C for 45 sec, ending with a final extension period of 10 at 72 C The PCR products were held at C until use The second stage of nested PCR was performed under the same reaction conditions by amplifying 3ml of the outer PCR product with inner primer pairs The temperature and time for annealing, denaturing, and exten-sion were the same Mock-infected cell culture fluids and porcine serum known to be negative for PRRSV and its an-tibody were included in all experiments as negative controls. For nested PCR, a nontarget control consisting of 22ml of reaction mix and 3ml of water was also included To avoid cross-contamination among samples, mainly with nested PCR, precautions were taken using separate rooms for re-agent preparation, manipulation, and detection of PCR prod-ucts and using separate supplies and pipettes, lab coats, and gloves in each room.
Detection of PCR products PCR products were detected
by gel electrophoresis in 1.5% agaroseein TAE buffer (0.04
M Tris-acetate, pH 8.5, with 0.002 M ethylenediaminetet-raacetic acid), stained with ethidium bromide, and visualized by photography with ultraviolet (UV) light. fX174 RF DNA/HaeIIIf was used as a molecular weight marker The
specificity of PCR products was confirmed by observing the expected size of the products on ethidium-bromide-stained gels In addition, specificity of PCR products from field isolates and reference isolates, amplified by P71/72, was also confirmed by Southern blotting.
cDNA probe labeling and Southern blot hybridization.
The internal oligonucleotide probe was labeled with digox-igenin-11-dUTP using a commercial oligolabeling kit.gThe
rapid alkaline method was used to transfer PCR products from denatured gels (0.4 N NaOH, M NaCl for 15 min) to nylon membranes.hAfter hr, the membranes were
neu-tralized and washed (0.5 M Tris, pH 7.2, M NaCl) in 3 washes each for with agitation, rinsed in water, and fixed on a UV transilluminator for Prehybridization was performed for hr at 42 C in 20 ml of prehybridization solution (53standard saline citrate [SSC], 1% w/v blocking reagent,g 0.1% N lauryl sarcosine, 0.02% sodium dodecyl
sulfate [SDS]) The membranes were hybridized for hr at 42 C in fresh prehybridization solution containing digoxi-genin-labeled probe in a final concentration of pmol/ml. After washing twice for in 23 SSC, 0.1% SDS and twice for 15 in 0.53 SSC, 0.1% SDC, 0.1% SDS, at room temperature, hybridization was detected as follows. Blocking was performed with 2% blocking reagent in 100 mM Tris-HCl, 150 mM NaCl, pH 7.5, for 30 min, and the membranes were incubated with anti-digoxigenin Fab frag-ments conjugated with alkaline phosphatasegdiluted in fresh
blocking buffer for 30 The membranes were then washed twice in 100 mM Tris-HCl, 150 mM NaCl, for 15 min and once in 100 mM Tris-HCI, 100 mM NaCI, 50 mM MgCI2, for After an overnight incubation in the dark
with 700ml of Lumi-Phos,gthe membranes were exposed to
X-ray film for various periods of time (15–30 min). Results
The expected size bands were observed in each PCR product from each primer pair in
ethidium-bromide-stained agarose gels Amplification of VR-2332 with all the primer pairs used is shown in Fig One of the reference strains (ATCC VR-2332) could be de-tected with all primers tested, but the Lelystad virus was amplified only with primers from ORF 1b (LV-1b) and with pair of primers from ORF (P71/72). However, the Lelystad virus could not be amplified with another primer set (P8214/8215) from ORF (Ta-ble 3) This result is in agreement with results of
pre-vious studies4 that used the same sequences but under
different reaction conditions.
All 24 field isolates and the reference strains could be detected by RT-PCR when primers from ORF 7 (P71/72) were used for amplification With primer sets from ORFs and (P41/42 and P61/431), 22 (92%) and 23 (96%) of the field isolates, respectively, were amplified The LV-1b primer was the least sensitive, detecting 21 of 24 isolates (88%) Nested PCR de-tected all of the isolates No amplification was ob-served with negative controls or RNA extracted from pseudorabies virus, transmissible gastroenteritis virus, and bovine viral diarrhea virus.
To determine if the presence of serum might inter-fere with PCR, field isolates and reinter-ference strains were diluted 1:5 in porcine serum and tested by PCR. Some of the primers (41/42, 61/431, LV-1b) failed to detect some of the isolates (IL-4, MN-1, MO-2, SD-1) in diluted serum (Table 4) However, these diluted isolates (with the exception of the Lelystad virus) were detected by the nested RT-PCR procedures used (Ta-ble 4).
(4)Table 3. Sensitivity of different primer pairs for the detection of PRRSV isolates
Isolate
PCR
41/42 71/72 61/431 LV-1b
Nested PCR-1 8214/8215 8216/8217 Nested PCR-2 61/72 71/431 IL-4 IL-6 IN-4 IA-1 IA-2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 IA-3 IA-15 IA-16 KS-1 KS-2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 KY-2 MN-1 MN-3 MN-4 SG-1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 SG-3 SG-4 SG-5 HL WL 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 MO-2 NE-1 OH-1 1 1 1 1 2 1 1 1 1 1 1 SD-1 VR-2332 Lelystad 1 1 1 2 1 1 1 1 1
Table 4. Effect of swine serum on the sensitivity of PCR for PRRSV detection The isolates were added to porcine serum known to be negative for PRRSV and its antibody Viral RNA from serum samples was extracted and amplified by PCR procedures
Isolate
PCR
41/42 71/72 61/431 LV-1b
Nested PCR-1 8214/8215 8216/8217 Nested PCR-2 61/72 71/431 IL-4 IA-1 KS-1 MN-1 MO-2 1 2 1 1 1 1 1 2 1 1 1 1 1 1 OH-1 SD-1 VR-2332 Lelystad 2 1 1 2 1 1 1 1 1
Southern blot hybridization Two of the isolates (SD-1, IL-4) did not hybridize, although sharp and bright bands were observed in ethidium-bromide-stained gels (Fig 2) The membranes were then stripped in 0.2 N NaOH and 0.1% SDS and reprobed at lower hybrid-ization temperatures of 37 C and 30 C There was no appreciable difference in the sensitivity of the hybrid-ization probe at these temperatures when compared with hybridization at 42 C (data not shown).
To investigate further, amplified products from SD-1, IL-4, and MN-3 were sequenced, and the sequence was aligned to that of VR-2332 The 20-nucleotide sequence corresponding to the sequence of the internal
probe was also compared Complete identity was ob-served between MN-3 and VR-2332 sequences, but nucleotide variability was observed in SD-1 and IL-4. The sequence of isolate SD-1 exhibited 15% (3/20) variability, and isolate IL-4 showed 10% (2/20) nucle-otide divergence when compared with the same region on VR-2332.
Discussion
(5)sev-Figure 2. Comparison of PCR detection from PRRSV isolates by agarose gel electrophoresis (A) and Southern blot hybridization using a digoxigenin-labelled oligonucleotide probe (B) Lanes 1–8: isolates IN-4, WL, MN-3, IL-4, IA-1, SD-1, OH-1, and ATCC VR-2332, respectively; lane MM: molecular weight markerfX174 RF DNA/HaeIII; lane ML: molecular weight marker XI labeled with digoxigenin.
eral regions of the viral genome The results of this study showed that RT-PCR can detect PRRSV RNA from field isolates but that the sensitivity of detection depends upon the region of the genome used for prim-er design Diffprim-erences in detection are probably due to strain variability; antigenic variability of these isolates
has been reported.2,8
The ORF is a robust target for diagnostic RT-PCR amplification; primers from this region allowed the de-tection of the corresponding fragment in all isolates tested The Lelystad virus could also be detected by 1 pair of primers from ORF (P71/72) but not with another from the same region (P14/P15) This infor-mation is useful in the design of studies whose objec-tive may either be to detect a broad range of PRRSV isolates or to determine strain differentiation.
The decreased sensitivity of some primers to some of the isolates diluted in serum may be related to losses in the RNA extraction procedure or degradation of RNA by contaminating ribonuclease present in the se-rum rather than to lower sensitivity of the primers be-cause the same primers could detect other isolates with
lower viral titers These experiments were repeated at least times, so the results not appear to be arti-facts Elsewhere, 2.5–4-fold losses from serum during
extraction procedures have been reported.26
The specificity of amplification was confirmed by including other common swine RNA viruses as neg-ative controls and by specific Southern blot hybridiza-tion No hybridization signals were observed in other RNA virus samples or in negative controls (data not shown) Confirmation of positive bands by nonra-dioactive chemiluminescent hybridization was easy and sensitive The convenience, simplicity, and spec-ificity of chemiluminescent detection have been
re-ported previously.1,9,24 The variability found in the
se-quence of isolates explains the lack of hybridization with the internal probe Oligonucleotide probes are short, and individual nucleotide changes of the target genomic sequence can influence the sensitivity of tection Genomic variability of PRRSV isolates
de-tected by RT-PCR has previously been reported.11,16
(6)oligonucle-otide probe hybridization to confirm the identity of PRRSV isolates for diagnostic purposes.
Acknowledgements
We thank Tom Molitor and Jane Christopher-Hennings for information on primer and probe sequences, Margaret Elam and Elida Bautista for oligonucleotide synthesis and helpful discussions, Dennis Foss for valuable technical assistance, and Judy Laber for DNA sequencing This project was fund-ed in part by the National Pork Producers Association and the Minnesota Agricultural Experiment Station.
Sources and manufacturers a QIAamp HCV, QIAGEN, Chatsworth, CA b Perkin Elmer Cetus, Norwalk, CT
c Dr J Christopher-Henning, South Dakota State University, Brookings, SD
d Ransom Hill Bioscience, Ramona, CA
e NuSieve agarose, FMC Bioproducts, Rockland, ME f GIBCO BRL, Gaithersburg, MD
g Boehringer Mannheim, Indianapolis, IN h Magnagraph, MSI, Westboro, MA
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